It is known in the art that it is possible to covalently link polyethylene glycol (PEG) polymers to therapeutic polypeptides, a process termed pegylation. Pegylation is normally carried out reacting the polypeptide with a reactive derivative of the PEG molecule, typically using a derivative that includes a functional group such as a hydroxyl, a thiol, an amine or a carboxylic acid group. In pegylation reactions, the reactive functional group of the PEG molecule forms a stable covalent bonds with amino, carboxy or thiol groups of amino acids present in the polypeptide, or with the reactive N- or C-termini of the polypeptide. In general, pegylation reactions require the use of a reactive cross-linking reagent such as maleimide.
However, it is also known that pegylation is a relatively uncontrolled reaction, mainly as a result of the use of the reactive cross-linker and because the PEG molecules can react in a number of different ways with multiple functional groups present in polypeptides, thereby producing heterogeneous products. This means that the resulting pegylated polypeptides can have linear or branched structures and/or have a variable number of PEG molecules coupled to each polypeptide. The reaction is also influenced by factors such as protein type and concentration, reaction time, temperature and pH value, all of which have an effect on the end products of the reaction.
In general, pegylation is carried out to influence the pharmacokinetic or immunological properties of polypeptides, especially those that are intravenously administered. In particular, the polypeptides can be altered to improve their stability, biological half-life, water solubility and immunologic characteristics. It is thought that pegylating a polypeptide to increase its mass (e.g., by 20-50 kDa) means that it is less readily excreted through the kidneys and therefore persists in the body for longer. In addition, pegylated polypeptides are thought to be protected from the degradation by endogenous enzymes, which also helps to increase their in vivo half-life. It is further thought that increasing the in vivo half-life and improving the efficacy of polypeptide drugs can lead to a reduction in the frequency or dose of the polypeptide that a patient requires, and hence help to reduce immune reactions against the polypeptide. Pegylation can also help to improve the water solubility to hydrophobic polypeptides.
However, while the properties of pegylated polypeptides are desirable, especially in therapeutic polypeptides, the lack of control in the pegylation reactions remains a problem as it often leads to a range of products having a range of different properties. Moreover, as polypeptides are complex molecules, it is difficult to use protecting groups to control the site and/or extent of the reaction, for example as would be possible in small molecule chemistry.
The glycosylation of polypeptides is a natural form of post-translational modification that alters the structure and function of polypeptides. In nature, glycosylation is introduced by an enzymatic process that leads to site specific modification of different types of glycosylated polypeptides. In N-linked glycosylation, glycans are attached to the amide nitrogen of asparagine side chains and in O-linked glycosylation, glycans are attached to the hydroxy oxygen of serine and threonine side chains. Other forms of glycosylation include glycosaminoglycans which are attached to the hydroxy oxygen of serine, glycolipids in which the glycans are attached to ceramide, hyaluronan which is unattached to either protein or lipid, and GPI anchors which link proteins to lipids through glycan linkages.
There is a general problem in the art in that glycosylation is often added to polypeptides in eukaryotic cells, but is rarely added to polypeptides expressed in the prokaryotic hosts often used for the recombinant expression of therapeutic polypeptides. This absence of glycosylation in polypeptides produced in prokaryotic hosts can lead to the polypeptides being recognised as foreign or mean that they have the properties that otherwise differ from their native forms. There is also a problem that it is difficult to engineer glycosylation into polypeptides at sites where there is not native glycosylation, in an attempt to use this to modulate the properties of the polypeptides.
WO 88/05433 discloses cross-linking reagents for producing conjugates of polypeptides and signal producing or cytotoxic chemical entities. The cross-linking reagents consist of a range of aromatic nitrogen-containing heterocyclic compounds, in particular 2-, 3- or 4-vinyl pyridines and vinyl pyrimidines, to which the signal producing or cytotoxic chemical entities can be attached via activated ester substituents present in the cross-linking reagent. The conjugation reaction relies on the vinyl groups of the reagents reacting with thiol groups present in the polypeptides. The compounds disclosed in WO 88/05433 all include an electron withdrawing substituent as a functional group of the aromatic nitrogen-containing heterocyclic ring. It is also notable that WO 88/05433 does not provide any examples that show the linkage of the signal producing or cytotoxic chemical entities to a polypeptide using the cross-linking reagents.
WO 2005/024041 describes a method of mass-tagging proteins in two or more cell populations for use in proteomics methods. The tagging involves reacting cysteine residues in one of the populations with 2-vinylpyridine, and in the other population with a C1-4 alkyl substituted 2-vinylpyridine, thereby producing tagged proteins with masses that are distinguishable by mass spectroscopy.
Banas et al (Biochimica et Biophysica Acta, 957(2): 178-84, 1988) studied the reactivity of cysteine residues of E. coli phosphofructo-1-kinase, using vinyl pyridine, bromopyruvate and dithionitrobenzoic acid to determine the relative reactivity of the six cysteine residues present in the polypeptide.
Fullmer (Analytical Biochemistry, 142(2): 336-9, 1984) describes a method of improving the analysis of the amino acid composition of peptides, in particular the identification of cysteine and tryptophan. One of the improvements uses 4-vinyl pyridine to protect half of the cysteine residues present in a peptide after reductive cleavage of disulphide bonds as the cysteine derivatives produced are stable to acid hydrolysis.
There remains a problem in the art in improving the derivatisation of polypeptides with coupling partners such as PEG molecules and glycan groups.